Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) tests that measure transcript abundance of selected genes in clinical specimens have been demonstrated to increase cancer diagnostic accuracy and enable “personalized medicine” through selection of the most effective treatment for each cancer.
There are challenges to using such methods, however, and these methods do not work well when the amount of sample is small or when the sample contains degraded RNA.
A particular challenge is that current clinical pathology sample collection and processing procedures focus on formalin fixation and paraffin embedding (FFPE) and fresh/fresh-frozen tissues rarely are available for molecular analysis. FFPE samples are difficult to work with because they yield RNA that 1) often contains PCR-interfering substances, and 2) is uniformly highly fragmented and often in low abundance. Economic factors prevent changing this workflow to ensure collection of samples in a form more conducive to molecular genetic analysis, such as fresh-frozen, therefore, there is a need to develop methods that are sufficiently robust to reliably conduct molecular genetic analysis in FFPE samples. These RT-qPCR tests must be able to assess FFPE samples with quality control and inter-laboratory reproducibility.